McDade H C, Cox G M
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Med Mycol. 2001 Feb;39(1):151-4. doi: 10.1080/mmy.39.1.151.154.
Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.
新型隐球菌是研究真菌感染分子发病机制的优秀模型系统。在该生物体的转化中，只有一种主要的可选择标记可被使用，而我们想要开发另一种。我们发现，新型隐球菌的各种菌株对氨基糖苷类抗生素制霉菌素非常敏感，并且对这种药物的自发抗性必定是极其罕见的事件。对制霉菌素的抗性由来自诺尔斯链霉菌的制霉菌素乙酰转移酶基因（nat1）的产物赋予。为了在新型隐球菌中表达该基因，我们通过用新型隐球菌肌动蛋白基因的启动子序列驱动nat1的表达创建了一个融合构建体。用该构建体对A型新型隐球菌菌株H99和D型菌株JEC21进行基因枪转化，分别产生了约1000个转化子/μg DNA和20个转化子/μg DNA的转化效率。使用一些H99转化子的DNA进行Southern印迹分析，这证实了所有抗性分离株都有构建体以随机方式整合到基因组中。制霉菌素抗性转化子对潮霉素B没有交叉抗性；潮霉素B是新型隐球菌中用作主要选择标记的另一种抗生素。开发制霉菌素抗性作为第二种主要的可选择标记将有助于对这种重要致病真菌的未来分子研究。
